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| Bovine κ-casein macropeptide (CMP), the C-terminal part of bovine κ-casein comprising 64 amino acid residues, plays a crucial role in maintaining the stability of the casein micelles in milk and also exhibits various biological activities and nutritive properties. CMP is quite heterogeneous consisting of numerous molecular forms due to a variety of post-translational modifications including both phosphorylation and O-glycosylation in addition to genetic modifications. The PTMs that are believed to affect the functional properties of CMP were investigated in this study. The aim was to develop a mass spectrometry-based strategy for the extensive characterization of CMP - discovery and site occupancy of phosphoylation and glycosylation as well as glycan structure elucidation. RP-HPLC separation of CMP not only led to isolation of individual subcomponents in CMP but also allowed structural assignments of 22 different molecular forms in combination with MALDI TOF MS analysis. After Glu-C digestion, different enrichment techniques such as TiO2 and ZIC-HILIC or other experimental approaches like neuraminidase treatment and β-elimination were ultilized prior to mass spectrometry for the recovery and enhanced detection of phosphopeptides and glycopeptides. The tetrasaccharide structure GalNAc(NeuAc)-Gal-NeuAc was determined as the main sugar chain attached to CMP by MALDI Q-TOF analysis. MSn analysis of phosphopeptides allowed the determination of four phosphorylation sites in the use of different instruments (MALDI Q-TOF, LC ESI Q-TOF and LTQ-FT), two of which are new. LTQ-FT MS analysis of intact glycopeptides with glycans attached allowed both the peptide sequence determination and the modified sites assignments. Moreover, LTQ-FT MS analysis of glycopeptides after β-elimination with ammonia also allowed the localization of the glycosylated sites. Four glycosylation sites were identified, at least one of which is new. The higher heterogeneity of CMP discovered in this study can give better idea on how this 'hairy' macropeptide functions in the maintenance of the equilibria between casein micelles and milk serum, make good sense in understanding the biological properties of CMP, and thus contribute to the food and pharmaceutical industries which are based on the activities and functions of CMP. |
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| Keywords:Post-translational Modifications; κ-casein macropeptide; phosphorylation; glycosylation; mass spectrometry |
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